Jostein Halgunset

TNF, IL-1, IL-6, IL-8 and soluble TNF receptors in relation to chorioamnionitis and premature labor

Inflammatory cytokines seem to play a key role in mechanisms initiating labor. Since cytokine levels are higher in preterm than in term labor, it has been hypothesized that labor-inducing effects of cytokines are inhibited by an upregulated production of cytokine antagonists, such as soluble cytokine receptors, at early stages of gestation. In this study, TNF, IL-1, IL-6, IL-8 and soluble TNF receptors (sTNFRs) were measured in amniotic fluid samples from a) 39 women in premature labor, b) 25 women who where not in labor but delivered prematurely, and c) 33 women in term labor. Fifty-four of the placentas from premature deliveries were evaluated for presence of histological chorioamnionitis. Chorioamnionitis was associated with increased levels of TNF, IL-1 and IL-6, whereas elevated IL-1, IL-6 and IL-8 concentrations were found in premature parturition with no signs of infection. Concentrations of sTNFR were lower in preterm than in term deliveries. The present study confirms the participation of inflammatory cytokines in parturition. Multivariate analysis suggests a dominant, role of IL-1 in the presence of chorioamnionitis, whereas IL-6 seems to be more important during idiopathic premature labor. TNFR data do not support the hypothesis that production of cytokine antagonists is upregulated prematurely to prevent partirution.

Histologic chorioamnionitis and umbilical serum levels of pro-inflammatory cytokines and cytokine inhibitors.

Objective To study 1. whether leucocyte infiltration in placenta tissues is associated with elevated umbilical serum levels of inflammatory mediators, and 2. whether leucocyte infiltration in the presence of neonatal disease is associated with additional increase in mediator levels.

Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner.

Paraffin-embedded tissue is an important source of material for molecular pathology and genetic investigations. We used DNA isolated from microdissected formalin-fixed, paraffin-embedded gastric tumors for mutation analysis of a region of the human gene for uracil-DNA glycosylase (UNG), encoding the UNG catalytic domain, and detected apparent base substitutions which, after further investigation, proved to be polymerase chain reaction (PCR) artifacts. We demonstrate that low DNA template input in PCR can generate false mutations, mainly guanine to adenine transitions, in a sequence-dependent manner. One such mutation is identical to a mutation previously reported in the UNG gene in human glioma. This phenomenon was not caused by microheterogeneity in the sample material because the same artifact was seen after amplification of a homogenous, diluted plasmid. We did not observe genuine mutations in the UNG gene in 16 samples. Our results demonstrate that caution should be taken when interpreting data from PCR-based analysis of somatic mutations using low amounts of template DNA, and that methods used to enrich putative subpopulations of mutant molecules in a sample material could, in essence, be a further amplification of sequence-dependent PCR-generated artifacts.

Cytokine levels in amniotic fluid and inflammatory changes in the placenta from normal deliveries at term

Cytokine levels in amniotic fluid have been shown to increase towards term in normal pregnancies, and may play a regulatory role in parturition by stimulating the local production of prostaglandins. The work reported in the present paper was conducted in order to test the hypothesis that the increased cytokine levels may be induced by a subclinical inflammatory reaction in intrauterine tissues. The concentrations of tumor necrosis factor (TNF), interleukin 1 (IL-1), interleukin 2 (IL-2) and interleukin 6 (IL-6) were determined in samples of amniotic fluid from 38 women in delivery at term, after a clinically normal pregnancy. In 33 of the cases, tissue material was available for histological examination. In these, the extent of inflammatory cell infiltration was assessed in the fetal membranes, placenta and umbilical cord. A close interrelation was observed between the levels of the mediators typically released during inflammatory processes (TNF, IL-1, IL-6). Frank chorioamnionitis was not found in any of the histological specimens, although most placentae showed varying degrees of granulocyte infiltration in the fibrin layer under the chorion, sometimes also in the chorionic membrane. The degree of such leukocytic infiltration correlated positively with the levels of TNF, IL-1 and IL-6. These findings lend support to the hypothesis that a low-level inflammatory process may be a normal occurrence in the term placenta, and that this process may induce the production of cytokines, which, in turn, may play a role in the regulation of parturition. Such inflammation could be due to exposure of the fetal membranes to microbial material from the vagina, as the cervix dilates towards term.

Spermine and Citrate as Metabolic Biomarkers for Assessing Prostate Cancer Aggressiveness

Separating indolent from aggressive prostate cancer is an important clinical challenge for identifying patients eligible for active surveillance, thereby reducing the risk of overtreatment. The purpose of this study was to assess prostate cancer aggressiveness by metabolic profiling of prostatectomy tissue and to identify specific metabolites as biomarkers for aggressiveness. Prostate tissue samples (n = 158, 48 patients) with a high cancer content (mean: 61.8%) were obtained using a new harvesting method, and metabolic profiles of samples representing different Gleason scores (GS) were acquired by high resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS). Multivariate analysis (PLS, PLS-DA) and absolute quantification (LCModel) were used to examine the ability to predict cancer aggressiveness by comparing low grade (GS = 6, n = 30) and high grade (GS≥7, n = 81) cancer with normal adjacent tissue (n = 47). High grade cancer tissue was distinguished from low grade cancer tissue by decreased concentrations of spermine (p = 0.0044) and citrate (p = 7.73·10−4), and an increase in the clinically applied (total choline+creatine+polyamines)/citrate (CCP/C) ratio (p = 2.17·10−4). The metabolic profiles were significantly correlated to the GS obtained from each tissue sample (r = 0.71), and cancer tissue could be distinguished from normal tissue with sensitivity 86.9% and specificity 85.2%. Overall, our findings show that metabolic profiling can separate aggressive from indolent prostate cancer. This holds promise for the benefit of applying in vivo magnetic resonance spectroscopy (MRS) within clinical MR imaging investigations, and HR-MAS analysis of transrectal ultrasound-guided biopsies has a potential as an additional diagnostic tool.

Changes in Gene Transcription Underlying the Aberrant Citrate and Choline Metabolism in Human Prostate Cancer Samples

Purpose: Low concentrations of citrate and high concentrations of choline-containing compounds (ChoCC) are metabolic characteristics observed by magnetic resonance spectroscopy of prostate cancer tissue. The objective was to investigate the gene expression changes underlying these metabolic aberrations to find regulatory genes with potential for targeted therapies. Experimental design: Fresh frozen samples (n = 133) from 41 patients undergoing radical prostatectomy were included. Histopathologic evaluation was carried out for each sample before a metabolic profile was obtained with high-resolution magic angle spinning (HR-MAS) spectroscopy. Following the HR-MAS, RNA was extracted from the same sample and quality controlled before carrying out microarray gene expression profiling. A partial least square statistical model was used to integrate the data sets to identify genes whose expression show significant covariance with citrate and ChoCC levels. Results: Samples were classified as benign, n = 35; cancer of low grade (Gleason score 6), n = 24; intermediate grade (Gleason score 7), n = 41; or high grade (Gleason score ≥8), n = 33. RNA quality was high with a mean RNA Integrity Number score of 9.1 (SD 1.2). Gene products predicting significantly a reduced citrate level were acetyl citrate lyase (ACLY, P = 0.003) and m-aconitase (ACON, P < 0.001). The two genes whose expression most closely accompanied the increase in ChoCC were those of phospholipase A2 group VII (PLA2G7, P < 0.001) and choline kinase α (CHKA, P = 0.002). Conclusions: By integrating histologic, transcriptomic, and metabolic data, our study has contributed to an expanded understanding of the mechanisms underlying aberrant citrate and ChoCC levels in prostate cancer. Clin Cancer Res; 18(12); 3261–9. ©2012 AACR.

A New Method to Provide a Fresh Frozen Prostate Slice Suitable for Gene Expression Study and MR Spectroscopy

Fresh frozen tissue from radical prostatectomy specimens is highly valuable material for research on gene expression and cellular metabolites. The purpose of this study was to develop a standardized method to provide a representative high quality research sample from radical prostatectomy specimens without interfering with the routine histopathological procedure.

Principal component analysis for the comparison of metabolic profiles from human rectal cancer biopsies and colorectal xenografts using high-resolution magic angle spinning 1H magnetic resonance spectroscopy

BackgroundThis study was conducted in order to elucidate metabolic differences between human rectal cancer biopsies and colorectal HT29, HCT116 and SW620 xenografts by using high-resolution magnetic angle spinning (MAS) magnetic resonance spectroscopy (MRS) and for determination of the most appropriate human rectal xenograft model for preclinical MR spectroscopy studies. A further aim was to investigate metabolic changes following irradiation of HT29 xenografts.MethodsHR MAS MRS of tissue samples from xenografts and rectal biopsies were obtained with a Bruker Avance DRX600 spectrometer and analyzed using principal component analysis (PCA) and partial least square (PLS) regression analysis.Results and conclusionHR MAS MRS enabled assignment of 27 metabolites. Score plots from PCA of spin-echo and single-pulse spectra revealed separate clusters of the different xenografts and rectal biopsies, reflecting underlying differences in metabolite composition. The loading profile indicated that clustering was mainly based on differences in relative amounts of lipids, lactate and choline-containing compounds, with HT29 exhibiting the metabolic profile most similar to human rectal cancers tissue. Due to high necrotic fractions in the HT29 xenografts, radiation-induced changes were not detected when comparing spectra from untreated and irradiated HT29 xenografts. However, PLS calibration relating spectral data to the necrotic fraction revealed a significant correlation, indicating that necrotic fraction can be assessed from the MR spectra.

Incorporation of contrast media in cultured cells.

Monolayer cultures of human prostatic (PC-3) and cervical (NHIK 3025) carcinoma cells were grown on formvar film and exposed to moderate concentrations of contrast agents for 30 minutes to 4 hours. After the exposure period, the monolayers were quickly frozen, and cryosections were examined by electron microscopy and X-ray microanalysis. Iodine was not detected in control cells, but was found in the cells that had been exposed to iodine-containing contrast media. The amount of intracellular iodine increased with increasing exposure dose and time. Because the cells mostly presented no sign of membrane damage, our findings support the view that contrast media have the ability to enter intact cells.

Inhibitors of tyrosine kinase inhibit the production of urokinase plasminogen activator in human prostatic cancer cells

Urokinase‐type plasminogen activator (uPA) seems to be an important protease in prostate cancer invasion, and tyrosine phosphorylation is thought to play a role in the regulation of its production. The amount of uPA was measured with a synthetic peptide substrate after treatment with various concentrations of tyrosine kinase inhibitors (TKI). The effect on proliferation and apoptosis was also assayed. Non‐toxic levels of genistein or the tyrphostin AG 490 produced up to 50% reduction of the uPA production in PC‐3 and DU‐145. The tyrphostins AG 1296 and AG 1478 inhibited uPA production in PC‐3 cells, whereas DU‐145 showed a slight increase of uPA production. TKI neither induced any detectable apoptosis, nor was there any reduction in proliferation rate. TKI can profoundly modify the production of uPA in prostatic cancer cells, thus indicating their possible use as suppressors of the invasive phenotype. The therapeutic potential of TKI warrants further investigation.